Alloxan-5-thiosemicarbazone as an antibacterial agent

ABSTRACT

ALLOXAN-5-THIOSEMICARBAZONE CAN BE USED TO INHIBIT AND/OR PREVENT THE GROWTH OF UNDESIRABLE BACTERIA, FUNGI AND FOLIAGE. THIS INVENTION IS PARTICULARLY CONCENTRATED WITH THE BACTERIOSTATIC AND BACTERICIDAL PROPERTIES OF ALLOXAN-5THIOSEMICARBAZONE AGAINST SPECIES OF ERWINIA, STAPHYLOCOCCUS, AND SALMONELLA.

United States Patent 3,773,952 ALLOXAN-S-THIOSEMICARBAZONE AS AN ANTIBACTERIAL AGENT John D. Douros, Jr., Littleton, Milan Brokl, Denver, and

Al F. Kerst, Littleton, Colo., assignors to The Gates Rubber Company, Denver, C010.

No Drawing. Filed July 6, 1971, Ser. No. 160,143 Int. Cl. A0111 9/00, 9/22 U.S. Cl. 424-251 '14 Claims ABSTRACT OF THE DISCLOSURE Alloxan-S-thiosemicarbazone can be used to inhibit and/or prevent the growth of undesirable bacteria, fungi and foliage. This invention is particularly concerned with the bacteriostatic and bactericidal properties of alloxan-S- thiosemicarbazone against species of Erwinia, Staphylococcus, and Salmonella.

BACKGROUND OF THE INVENTION The chemical literature reveals few subjects, even within the rapidly expanding discipline of biochemistry, which have generated so many patent applications as the areas involving the biochemical uses of compounds which contain the barbituric acid nucleus,

where R represents various substituents. Representative biochemical uses of such compounds are found in US. Pats. 2,561,688; 2,605,209; 2,725,380; 2,820,035; 2,887,- 487; and 3,102,072. However the antibacterial, antifungal and defoliant properties of alloxan-S-thiosemicarbazone have not previously been discovered.

SUMMARY OF THE INVENTION According to the present invention, it has been found that alloxan-S-thiosemicarbazone I II when present in effective quantities in a very effective cotton defoliant, as well as a bactericidal, and fungicidal agent.

The alloxan-S-thiosemicarbazone used in the present invention was prepared according to the following technique:

wherein 8.0 gr. (0.05 mole) of alloxan monohydrate, see, W. Hartman and O. Sheppard, Org. Syn. Coll., vol. IH, 37 (1955); J. Sperand and T. Daboirch, Org. Syn. Coll., vol. III, 39 (1955); and A. Holmgren and W. Wenner, Org. Syn. Coll., vol. IV, 23 (1963), and 4.56 gr. (0.05 mole) of thiosemicarbazide (Sigma C0. grade) are treated with 250 ml. of a water-ethanol mixture (1:1) in a 500 ml. flask. The mixture is refluxed in a water bath for 30 minutes. A yellow precipitate of the condensation product forms in a few seconds. After the reaction is substantially complete the reaction mixture is cooled and the precipitate separated on a sintered glass funnel, washed with small portions of water several times and finally washed with ethanol. It is then dried in a desiccator under vacuum for 2-3 hours. The yield is 10.6 gr. (98.5% of theory).

DESCRIPTION OF THE PREFERRED EMBODIMENTS Example I.-Antibacterial and antiyeast activity The in vitro effectiveness of alloxan-S-thiosemicarbazone against bacteria and yeast species is established in the following manner. One loopful of each of the investigated bacteria or yeast is transferred from agar slants to 10 ml. of trypticase soy broth and incubated at 37 for 18 hours. At the end of this period, the bacteria or yeast is seeded into the same medium (LS-2% agar) in which the original inoculum was prepared. The bacteria is then seeded at 1 ml. of inoculum per 250 ml. of medium, which is equivalent to at least 1 10 cells/ml. as determined by dilution platecount or nephelometer readings. The resultant mixtures are poured into heat-resistant sterile petri dishes at a temperature of 45 C. Analytical filter paper discs 1.2 cm. in diameter are used for the agar diffusion technique. Each disc is saturated with 0.08 ml. of the solubilized alloxan-S-thiosemicarbazone compound g./disc) in a suitable carrier and placed on the surface of the hardened agar. The plates are then incubated at 37 C. for 18 hours. The activity of the alloxan-S-thiosemicarbazone compound is established by measuring the zone of inhibition in centimeters. Untreated control plates are used as a basis for comparison and these exhibit a profuse growth of bacteria. The results of these tests are See footnote at end of table.

TABLE Cntlnued Zone of inhibition in centimeters Gram bacteri Shigella boydii-ATCC N0. 9212 Shigella boydii-ATCC6lgI0. 9905- Shtgella fleznen Type 6MMV 760.. Shigella flezneri Type iq-MMV 6625... Shigella dysenteriae Type 2-MMV 6673....

Salmonella sp.ATCC No. 7378..-. 1 Salmonella paralyphi-ATCC N o. 9 Salmonella enteritis-ATCC N 0. 13076 Salmonella pullommATCC No. 1039 Salmonella derb'J.ATOC N0. 6960...- Salmonella gallinarium-ATCC No. 9 lla t1]; ium-S R-11 Salmonella typhosa-ATGC No. 19403.-.--

Nez'sserz'a gonorrhoeae-ATCO No. 19424... Nez'sserla intracellularzs Neisseria meninaitides-ATCC N 0. 13077..

Listeria monocytogener-ATCC No. 15813- Vibrz'o felns-ATCC No. 15296.--.: Vibrio cholerae-ATCC No. 14035- Proteus valgaris--ATCC No. 4984 Erwinia carotavora-ATOG No. 495

Mycgiigeterium .tmegmatz's-ATCC No.

11 Mycobacterium fortuitum, Debos-ATCC No. 4243 Myeobaeterium atrium-ATOG No. 19421-.- Mycobacterium DOUi-S ATCC No. 19274 Mycobacterinm phlez-ATCC No. 11782,

phage host Micrococeus tetragena-ATCG No. 10875.-- Microcoecus melz'tensis-ATCO No. 19399.. Micrococcus lysodeikticus-ATCC N0. 4698.

Coryngbacterinm diphlheriae-ATCO No.

C'orynebaclerium haemolyticnm-ATCC N o. 9345 positive and Gram negative a 100, 10, r g/disc rig/disc a re-H rr r-rra see c rlo QoOOrsOOO cococ as COO o omen mqoomtoqom- 0 cos 0 one so oflwqwo oocoo Diplococcus pneumoniae-ATCC No. 6303. Hemophflus hemolyticus-ATCC No. 10014 Hemophilus influenzea-AICC No. 19418. Hemophilus parainfluenzae-ATCC No. 7901 Hemophilus ants-ATCC N 0. 19417 Hemophilus vaainalis-ATCC N 0. 14018.. Pseudomonas aeruainosa-ATCC No.

NNN 8. woo 01-- Pseudomonas aeruginosa-ATCC Psendomonaa aeruyinosa-ATCC No. 12055 Psendomonas aeruginosa28 Psendomtmas maltophiha-G 107. Pseudomonas-K997... Pseud0monas-K966 1 Hazy zone.

N orE.-tr= Trace.

This data indicates that alloxan-S-thiosemicarbazone compounds can be used to inhibit many important types of diseases. For example, they can be used against:

Bacteria: Disease produced Staphyloqoccus aureus Major cause of bovine mastitis. Streptococcus faecalis Bacterial endocarditis.

Escherichia coli Cause of scours and secondary cause of bovine mastitis. Proteus vulgaris Major cause of fish disease Pseudomonas aeruginosa The demonstrated antibacterial activity of alloxan-S- thiosemicarbazone compounds against Staphylococcus aureus and Escherichia coli is of particular interest to the field of pharmacology since the disclosed activity indicates that these compounds can be formulated as powders, salves, and ointments for administration in the treatment of burns and bacterially induced inflammations such as abscesses, dermatitis, rashes and the like, particularly in domestic animals.

Although the precise mode of action whereby alloxan-S- thiosemicarbazone inhibits bacteria growth is not completely understood, it is believed that the alloxan-S-thiosemicarbazone compounds of this invention may serve as chemical antagonists; that is, as chemicals which compete with enzymes essential to the development of many bac teria. Since enzymes perform their catalytic function by virtue of their affinity for their natural substrate; any compound resembling a substrate in its chemically critical aspect may also have an ailinity for the enzymes. If this afiinity is great enough, the analog will displace the normal substrate from the enzyme and will prevent the growth reaction from taking place. It is believed that alloxan-S-thiosemicarbazone has a chemical afiinity for an essential site one one enzyme necessary for bacterial growth and life.

The alloxan-S-thiosemicarbazone formulations of this invention can also contain other therapeutically valued supplements such as local anesthetics, irradiated oils, and other medicinal substances. When used for these or simi lar purposes, alloxan-S-thiosemicarbazone may be incorporated in any therapeutically acceptable carrier such as oils, salves and ointments, together with adjuvants comprising surface active agents, detergents, dispersing agents, stabilizers and other modifiers which may facilitate the handling and application of the antibacterial material. In the case of the in vitro applications of the compositions of this invention, it is diflicult to predict with precision what in all cases will constitute a therapeutic dose even on a weight basis. Variable factors such as type, duration and severity of infection and mode of administration may be determining factors for the establishment of therapeutic doses.

Those skilled in the art will recognize that the above data indicates that the scope of this invention should not be limited to any particular disease species or to any particular type of animal or plant life. For example, the noted activity of alloxan-S-thiosemicarbazone against Erwim'a carotovora suggests that this compound will also prove to be of value against such other Erwinia species as Erwinia amylovora, Erwinia atroseptica and Erwinia vitivora. Erwinia species are known to cause diseases of apples, carrots, tobacco, potatoes and squash.

Example HProtection of apple seedlings against Erwinia carotovora The test procedures used to establish the efficacy of alloxan-S-thiosenficarbazone against Erwinia carotovora, are as follows. MacIntosh apple seedlings in the true-leaf stage, grown from seed under greenhouse conditions are mounted on a compound turntable and sprayed at 30 pounds pressure with an alloxan-S-thiosemicarbazone solution at the concentrations indicated below. The alloxan-S- thiosemicarbazone is prepared for spraying by dissolving it in a suitable solvent such as acetone, methyl alcohol or ethyl alcohol and then diluting it to the desired concentration with deionized water containing wetting and dispersing agents.

After the treated seedlings have dried, they were placed among diseased Erwz'nia carotovora apple seedlings, and subjected to an initial spore shower by dusting with spores from diseased plants and then left undisturbed in place for approximately ten days. By this procedure treated plants are subjected to the cited initial spore shower as well as to continuing natural infection pressure from the surrounding inoculum. Observations ten days after initial inoculation are used to determine the duration of effec tiveness of treatments.

Untreated controls reflected a 75-100% leaf area distimeters. Untreated control plates are used as a basis for comparison and these exhibit a profuse growth of bacteria. The results of these tests are as follows:

eased at this time. The effectiveness of treatment was 5 Zone ofinhibition in determined by direct comparison with untreated inocucentlmeters lated controls. Captan was used as a reference standard. m ,9 9 32 The results of these tests are as follows: lemma Sm Aspergt'llus niger-ATCC No. 1004..-. 3. 9 2. 0 1. 1 Alternaria solani-ATCC No. 6396.. 4. 0 1. 6 1. 0 Alloxan-5-thlosemlearbazone: Rhizopus stolom'jer-ATCC No. 1040 4. 7 3. 8 1. 7 Concentration, p.p.m. 100 750 Fusarium ozysporum-UFCC 1l22 1. 0 1. 2 tr Percent control 82 100 Candida aZbican8S RI 523 3.8 2. 1 1. 1

Example III-Antifungal activity Example VSocondary fungal screen The antifungal activity of alloxan-S-thiosemicarbazone 5 A Secondary screefl using the tcchniques of mp compounds is established by treating Fusarium oxysporum, Produces the Q F at the alloxall-5-thloseml- Fusarium roseum, Rhizopus nigricans, Rhizopus sto lonicafbalone concentratlolls mdlcated! fer, Aspergillus niger and Alternaria solani test fungi in the following manner: One loopful of each of the tested Concentration viable fungi cultures, spores and mycelia is transferred 1 000 100 10 from an agar slant to an 80 ml. portion of the nutrient Microorganism p i/diso o /dis'o /diso broth composed of oatmeal agar, Czapeks, Sabouraud Rhodotonnasp Duke 4.1 M 21 and deionized water to volume. The 80 ml. portion of lFthizopusstoloniferqjkljloCcGlgg.10404. 1. 1 usartum TOSCILM- l the fungi and broth is then placed in a sterile shake flask MSW-um oxyspomm plus MWWUFCC (300 m1.) and the flask 1s placed on a rotary shaker for 25 1122 0.9 1.2 tr 96 to 120 hours at room temperature. At the end of this 2222235 2 2 .3 2 g" g g g incubation time period, 10 ml. of the liquid are homogfis er tgus ni h- 1017 gs; a 8 ergt 11/881] 0101- u enlzed and placed 111 another sterile shake flask (300 ml.) Asgemms MMMWATCC N0, 100% 39 20 L1 containing 80 ml. of the above nutrient broth and 60 As er zzos omts-A'roo No.9643 3.1 1.5 tr p.p.m. of alloxan-S-thiosemicarbazone. The flasks are 3;; 31 placed on a rotary shaker operating at 240 r.p.m. at room temperature for 3 to 9 days. After this second incubation time, the flasks are taken off and examined for visible Example II-Cotton defohant Propertles fungal growth and mycehal weights are determined. Un- Alloxan-S-thiosemicarbazone cotton defoliant propertreat d c tr ls a d as the s s of pans n and 35 ties were established by tests which indicate the following these display profuse fungal growths containing species efli a t the concentrations indicated. of Fusarium, Rhizopus, Aspergillus and Alternaria. The results of these tests indicate that the alloxan-S-thiosemi- Concentration lbs lam 1o 5 y oarbazono compounds of this invention p t a stan- Percent detonation.. Z:::::::::::::::::: 100 75 t2 tial degree of inhibition of fungal growth at 60 p.p.m. The results of these tests were as follows:

It Wlll also be recognlzed by those skilled in the art Fungi: Control at 60 p.p.m., percent that other protectant, systemic and eradicant procedures Fusarlum 42 may provide detection of other biological activities. Rhrzopus 48 Pathogens representative of Phycomycetes, Ascomycetes, Aspergillus 56 Basidiomycetes and the Fungi imperfecti may provide in- Alternarla 37 dices of other fungicidal activity. Additional pathogens and appropriate host organisms may well afford other Example IV Ant1funga1 and annyeast actlvlty opportunities to further define the degree and spectrum To further define the antifungal activity of alloxan-S- 50 of the activity disclosed in this invention. Since no firm thiosemicarbazone seeded agar plates are prepared by rules of procedure can be laid down for the sequence of transferring the cultures from slants washed with saline such evaluations or for the choice of pathogens, alloxan-S- or phosphate buffers to the surface of hardened Sabouraudthiosemicarbazone must be considered on the basis of its Dextrose agar plates. Again, as in the case of Example 1, demonstrated performance in such primary evaluations the alloxan-S-thiosemicarbazone is tested by impregnating and then progressively judged in subsequent studies. A filter paper discs (1.27 cm. in diameter) with 0.08 ml. wide range of pathogens, representative of economically of the solubilized alloxan-S-thiosemicarbazone compounds important diseases, can be used to help define alloxan-S- (100 ,ug./disc) and placing them on the surface of the thiosemicarbazones biological activity and to assure high hardened agar. The plates are then incubated at 30 C. degrees of success under field conditions. The following for 1 8 hours. The activity of alloxan-5-thiosemicarbazone disease organisms, crops and reference standards may be is established by measuring the zone of inhibition in cenused in such evaluations:

Disease Disease organism Reference compound Powdery mildew of cucumbers Erysiphe cichomcearu Maneb. Karathane. Leaf rust of wheat P rubzgo-cera..- D0.

D0 P rubigo-vera. Plantvax. Ri bl t i" Piriculart'a oryzae Blasticidin. Downy mildew of sugar beet Penmoapora schactii. Karathaue. Downy mildew of lime bean Phzltophthora phaseolL. 0. Bean rust. U1 var. 11/ Maneb. Powdery mildew of wheat Erysiphe gramintum..- Karathane. Powdery mildew of apple Poliosphaem leucotricha Do. Powdery mildew of roses. Sphaerotheca pamtosa v Do. Powdery mildew of eantalo Erysiphe cichoraceamm Do. Leaf spot of whea... Helminthosporium sativum. Maneb. Early blight of tomat Alternart'a solam' Do. R l t; ai na to Pt'ricularia oryzae" Blasticidin. Oerocspora leaf spot of sugar beets Cercospora bett'cola... Maneb. Septoria, leaf spot of celery Sepwriu upii-araveolentts. Do. Apph, qmzh Venturia inaequalis Oyprex. Common bacterial blight of b Y p Streptomycin sulfate.

Wherever possible, the applicants recommend the use of in vivo procedures to test the alloxan-S-thiosemicarbazone compositions of this invention to demonstrate their efficacy under more realistic conditions. However, not all pathogens lend themselves to such techniques. In Order to provide additional spectrum definitions, the following fruit-rotting, storage decay and bacterial pathogens may be tested by in vitro methods:

Brown rot of stone fruits Sclerotinia fructicola Grey mold of fruit and vegetables Botryz's cinerea Rhizopus fruit and vegetable rot Rhizopus nigrz'ca'ns Citrus green "mold P di Bacterial disease of many fruit crop P syring Bacterial soft rot Erwinia carotovora In their plant protection aspects, the alloxan-S-thiosemicarbazone compounds of this invention may be used in the manner known to the organophosphorus crop protection art; that is, they can be made up in solid or liquid formulations. Examples of solid formulations are dust, wettable powders, granules and pellets. As a dust, alloxan- S-thiosemicarbazone compounds may be dispersed in powdered solid carriers such as talc, soaps, soapstone, attapulgus clay as well as other finely divided solids known to the dusting art. When formulated as wettable powders, the active component may be employed in conjunction with inert fillers which may be of the clay type carrier or non-clay type, in conjunction with various combinations of wetting agents and emulsifiers which permit the adaptation of the concentration as a free-flowing powder for dispersion in the field.

Each of these carriers may in turn contain other carriers or extenders which are ordinarily non-reacting or inert substances such as sand, clays, talc, sawdust, alkaline earth carbonates, oxides, phosphates and the like as well as diatomaceous earth, micas or other suitable materials. When liquid formulations are desired, liquid extenders, dilutants or carriers of a non-reactive nature may be utilized. Examples of such materials are alcohols, ketones, glycols, aromatic hydrocarbons, petroleum fractions such as octane and various other distillates. From these considerations, it will also be recognized that the above formulations with slight modifications may be used in the field of animal husbandry as dusting powders and salves.

Where it is desired to use the aforementioned wettable powders or liquid formulations, either emulsified, dispersed or suspended in water or other fluids, one or more of the class of materials herein referred to as adjuvants can also be incorporated into the powder, dust or liquid formulation. These adjuvants comprise surface active agents, detergents, wettable agents, stabilizers, dispersing agents, suspending agents, emulsifying agents, spreaders, stickers and conditioning agents generally. To their modifying characteristics these adjuvants may facilitate handling and application and frequently enhance or potentiate the alloxan-S-thiosemicarbazone compositions of this invention in their biological activities by mechanisms which are frequently not well understood. A satisfactory but not exhaustive list of these adjuvants appears in Soap Chemical Specialties, vol. 31, No. 7, p. 61; No. 8, pp. 38-61; No. 9, pp. 52-67; and No. 10, pp. 38-67 (1955). See also, Bulletin No. 607 of the Bureau of Entomology and Plant Quarantine of the United States Department of Agriculture.

An additional advantage of alloxan-S-thiosemicarbazone is its compatibility with a variety of other bactericidal and fungicidal materials. For example, it may be convenient to combine alloxan-S-thiosemicarbazone with one or more other adjuvants, carriers, pesticides, biocides, or fungicides of various structures. For example, alloxan-S- thiosemicarbazone fungicidal inhibitors may be combined with insecticidal materials such as chlordane, benzene hexachlorides, DDT, DDD, and insecticidal carbamates, polychlorinated terpenes, parathions, methoxychlor, in-

Captan. Maneb.

Do. Kai-ethane. Captan.

organism, and side effects. Also to be considered is the cost of production and the characteristic solubility of al loxan-S-thiosemicarbazone in the carrier material.

The broad spectrum of antifungal activity afforded by alloxan-5-thiosemicarbazone can also be utilized in the formulation of disinfectant solutions, paints, coatings, films and polymeric materials in order to protect against disease and rot caused by various fungi species. When used as a disinfectant, suitable formulations may be prepared by mixing the compound with an emulsifying agent in the presence of organic solvents and then diluting it with water to form an aqueous emulsion containing the alloxan-S-thiosemicarbazone. Suitable emulsifying agents include, e.g. alkylbenzenesulfonates, polyalkene glycols, et cetera. Aqueous emulsions of alloxan-S-thiosemicarbazone are particularly suited for use in disinfectant solutions used in washing hospital floors and walls. The following examples further illustrate the antifungal properties of alloxan-S-thiosemicarbazone.

Example VII-Preparation of a vinyl coating resistant to mildew deterioration A vinyl coating is prepared using a commercially available preparation without the fungal growth inhibitor.

An identical vinyl coating is prepared except that 2% by weight of alloxan-J-thiosemicarbazone is incorporated into the coating formulation.

Two sets of components such as asbestos tubing, silkwrapped transformers and rayon-wrapped solenoids are obtained. ,One set is sprayed with the vinyl coating containing inhibitor, the other with the identical coating without inhibitor.

Example VHIPreparation of plasticizers resistant to mildew A commercial thermoplastic monomer is divided into portions which are treated as follows.

Portion 1: To the first portion is added 2% by weight of alloxan-S-thiosemicarbazone and 10% by weight of dimethylnaphthalate as plasticizer. The monomer is polymerized and molded into 3-inch diameter discs, A inch in thickness prior to testing.

Portion 2: To this portion is added 2% by weight of alloxan-S-thiosemicarbazone and 10% by weight of butyl isodecylphthalate as plasticizer. The monomer is polymerized and molded as above.

Portion 3: The portion is the untreated control of Portion 1 containing no fungal inhibitor but 10% by Weight of dimethylphthalate as plasticizer. Again, the polymerization and molding are identical.

Portion 4: This portion is the untreated control of Portion 2 containing no fungal inhibitor but 10% by weight of butyl isodecylphthalate as plasticizer. The polymerization and molding are as described above.

The two plasticizers were chosen on the basis of their known susceptibility to Fusarium attack under high humidity and temperature conditions.

Example IX--Vinyl coatings and plasticizers for fungal resistance The vinyl coated articles and controls of Examples VII and VIII are placed in an air-tight high temperature and high humidity chamber maintained at 80 F. and 95% humidity to simulate tropical temperature and humidity conditions. After a months exposure the vinyl coated articles treated with inhibitor are only slightly at-, tacked by rot while the articles coated with vinyl without the alloxan-S-thiosemicarbazone are well rotted. The two untreated control polymer discs are examined and found to be blackened and mildew rotted. Isolates of Aspergillus, Fusarium and known species of yeasts are prepared from the deteriorated discs. The discs containing the alloxan-S- thiosemicarbazone fungal inhibitor are not adversely affected.

Alloxan --thiosemicarbazone can be made up in solid or liquid formulations. Examples of solid formulations are dust, wettable powders, granules and pellets. Solid formulations, particularly floating solid formulations. As a dust, alloxan-S-thiosemicarbazone compounds may be dispersed in powdered solid carriers such as talc, soap, soapstone, attapulgus clay, as well as other finely divided solids. When formulated as wettable powders, the active alloxan-S-thiosemicarbazone component may be employed in conjunction with inert fillers which may be of the clay type carrier or non-clay type in conjunction with various combinations of wetting agents and emulsifiers which permit adaptation as a free flowing powder. Each of these carriers may in turn be combined with other carriers which are ordinarily non-reacting or inert substances such as sand, clays, talc, sawdust, alkaline earth carbonates, oxides, phosphates and the like, as well as diatomaceous earth, micas or other suitable materials.

When liquid formulations are desirable, liquid extenders or carriers of a non-reactive nature may be utilized. These compositions should contain approximately 0.1% to 20% by weight and preferably 0.1% to 3% and most preferably 0.5% to 2% of the active alloxan-S-thiosemicarbazone ingredient. Solvents which may be used in the preparation of such compositions would include alcohols, ketones, glycols, mineral spirits and aromatic solvents such as benzene, xylene, nitrobenzene, dimethylformide. Furthermore, to assist in the rapid and complete dispersion in water systems, these compositions may also contain approximately 5% to 30% by weight and preferably to by weight of surface-active agents. Suitable surface-active agents include sodium dialkyl sulphates, sodium alkylbenzene sulfonates, sodium carboxylates and the non-ionic surfactants such as ethoxylated fatty acid alcohol and amines.

Alloxan-S-thiosemicarbazone is compatible with a wide variety of other bactericidal and fungicidal materials. For example, it may be convenient to combine compositions with one or more of the other bactericides or algaecides. Common fimgicides and bactericides such as sulphur, inorganic salts such as copper sulphate, activated colloidal silver compounds, copper naphthenates and zinc acetate, as well as substituted hydrocarbons and quarternary ammonium compounds, may be employed.

Having thus disclosed our invention, we claim:

1. A method of killing bacteria of the genus Coryne-.

bacter which comprises applying to a plant infected with 10 said bacteria, a bacteria killing amount of alloxan-S- thiosemicarbazone.

2. A method of killing bacteria of the genus Micrococci, which comprises applying to a plant infected with said bacteria, a bacteria killing amount of alloxan-S-thiosemicarbazone.

3. A method of killing bacteria of the genus Mycobacterium which comprises applying to a plant infected with said bacteria, a bacteria killing amount of alloxan- 5-thiosemicarbazone.

4. A method of killing bacteria of the genus Diplococci which comprises applying to a plant infected with said bacteria killing amount of alloxand-thiosemicarbazone.

5. A method of killing bacteria of the genus Streptococci which comprises applying to a plant infected with said bacteria, a bacteria killing amount of aloxan-S-thiosemicarbazone.

6. A method of killing bacteria of the genus Shigella which comprises applying to a plant infected with said bacteria, a bacteria killing amount of alloxan-S-thiosemicarbazone.

7. A method of killing bacteria of the genus Salmonella which comprises applying to a plant infected with said bacteria, a bacteria killing amount of alloxan-S- thiosemicarbazone.

8. A method of killing bacteria of the genus Neisseria which comprises applying to a plant infected with said bacteria, a bacteria killing amount of alloxan-S-thiosemicarbazone.

9. A method of killing bacteria of the genus Hemophilus which comprises applying to a plant infected with said bacteria, a bacteria killing amount of alloxan-S- thiosemicarbazone.

10. A method of killing bacteria of the genus Pseudomonas which comprises applying to a plant infected with said bacteria, a bacteria killing amount of alloxan-S-thiosemicarbazone.

11. A method of killing Staphylococcus aureus which comprises applying to a plant infected with said Staphylococcus aureus a killing amount of alloxan-S-thiosemicarbazone.

12. A method of killing Xanthomonas phaseoli which comprises applying to a plant infected with said Xanthomonas phaseoli a Xanthomonas phaseoli killing amount of alloxan-S-thiosemicarbazone.

13. A method of killing Escherichia coli which comprises applying to a plant infected with said Escherichia coli an Escherichia coli killing amount of alloxan-S- thiosemicarbazone.

14. A method of killing Erwinia carotovom which comprises applying a plant infected with said Erwinia carotovora an Erwinia carotovora killing amount of alloxan- 5-thiosemicarbazone.

References Cited Chemical Abstracts, vol. 60, item 6844h-6845a, 1964.

ALBERT T. MEYERS, Primary Examiner D. W. ROBINSON, Assistant Examiner US. Cl. X.R. 424254 

